Ribosome—Inactivating Proteins
It has been known for a long time that extracts from many plant tissues possess anti-viral activity, which in several cases is due to proteins identified as inhibitors of protein synthesis, called ribosome-inactivating proteins (RIP, reviewed by Barbieri et al., Biochim. Biophys. Acta 1154:237 (1993)). The pokeweed anti-viral protein (PAP) was the first anti-viral protein to be identified as a RIP (reviewed by Irvin, in Antiviral Proteins in Higher Plants 65 (1994)). Subsequently, all other RIP tested possess anti-viral activity not only against plant viruses, but also against animal viruses, including HIV (reviewed by Battelli and Stirpe, in Antiviral Proteins in Higher Plants (1994)).
All RIP, either single-chain (type-1) or two-chain (type-2), enzymatically release adenine from a single nucleotide in a precise position (A4324 in the case of rat liver 28S rRNA, A2660 of E. coli rRNA) in a universally conserved GAGA tetraloop of the major rRNA (Endo and Tsurugi, J. Biol. Chem. 262:8128 (1987); reviewed by Barbieri et al., Biochim. Biophys. Acta 1154:237 (1993)). Depurinated ribosomes become unable to elongate the nascent peptide chain.
The anti-viral activity of these proteins was commonly attributed to the inactivation of ribosomes, with inhibition of protein synthesis of the host cell and consequent arrest of viral replication. However a degradation of supercoiled DNA in the presence of RIP was reported (Li et al., Nucleic Acid Res. 22:6309 (1991); Ling et al., FEBS Lett. 345:143 (1994); Roncuzzi and Gasperi-Campani, FEBS Lett. 392:16 (1996)). Moreover, at least some RIP release more than one adenine residue from ribosomes (Barbieri et al., Biochem. J. 286:1 (1992)) and act on RNA species other than ribosomal, including viral RNAs, on poly(A), and on DNA (Barbieri et al, Nature 372:624 (1994), Nucleic Acid Res. 25:518 (1997); Stirpe et al., FEBS Lett 382:309 (1996)). Thus many, if not all, RIP have polynucleotide;adenosinc glycosidase activity, which may have a role in the anti-viral activity besides the inactivation of the host cell ribosomes.
Immunotoxins
Immunotoxins are chimeric molecules in which cell-binding ligands are coupled to toxins or their subunits. The ligand portion of the immunotoxin is usually an antibody that binds to selected target cells. The toxin portion of the immunotoxin can be derived form various sources. Most commonly, toxins are derived from plants or bacteria, but toxins of human origin or synthetic toxins (drugs) have been used as well. Toxins used for immunotoxins derived from plants or bacteria all inhibit protein synthesis of eukaryotic cells, The most widely used plant toxin, ricin, consist of two disulfide-linked polypeptides A and B (Olsncs et al., in Molecular Action of Toxins and Viruses 51 (1982)). Another group of plant-derived toxins used in immunotoxins are the type-1 RIP. These molecules are single-chain proteins found in plants and have similar enzymatic properties as the A-chain of ricin (reviewed in Stirpe and Barbieri FEBS Lett. 195:1 (1986)).
The cross-linker used to join the ligand (antibody) and the toxin must remain stable when extracellular, but labile when intracellular, so that the toxin fragment can enter the cytosol. The choice of cross-linker depends on whether intact toxins, A-chains or type-1 RIP are used. A-chains and type-1 RIP are generally coupled to the ligand using linkers that introduce a disulfide blind between the ligand and the A-chain (Myers et al., J. Immunol. Meth. 136:221 (1991)). Intact toxins are usually linked to ligands using non-reducible linkages (such as thioether) to prevent release of the active free toxin in Vivo. Recombinant immunotoxins have been prepared by splicing the genes encoding the toxin to the gene encoding the ligand (for instance a recombinant antibody fragment) and expressing the entire immunotoxin as a fusion protein (Pastan et al, Ann, Rev. Biochem. 61:331 (1992)). Recombinant immunotoxins are highly stable in vivo because they contain non-reducible peptide bonds.
Various types of immunotoxins directed against different cellular targets have been evaluated in vivo, both in animal models and in phase I or II clinical trials. The results of a number of these studies are reviewed in Ghetie and Vitetta Curr. Opin. Immunol. 6:707 (1994) and Thrush et al., Ann. Rev. Immunol. 14:49 (1996).